title: A cytometric bead assay for sensitive DNA detection based on enzyme-free signal amplification of hybridization chain reaction.
abstract: A versatile flow cytometric bead assay (CBA) is developed for sensitive DNA detection by integrating the advantages of hybridization chain reaction (HCR) for enzyme-free signal amplification, flow cytometry for robust and rapid signal readout as well as magnetic beads (MBs) for facile separation. In this HCR-CBA, a biotinylated hairpin DNA (Bio-H1) is firstly immobilized on streptavidin-functionalized MBs. Upon the addition of target DNA, each target would hybridize with one Bio-H1 to open its hairpin structure and subsequently initiate a cascade of hybridization events between two species of fluorescent DNA hairpin probes (H1*/H2*) to form a nicked double helical DNA structure, resulting in amplified accumulation of numerous fluorophores on the MBs. Finally, the fluorescent MBs are directly analyzed by flow cytometry. This technique enables quantitative analysis of the HCR products anchored on the MBs as a function of target DNA concentration, and analysis of each sample can be completed within few minutes. Therefore, the HCR-CBA approach provides a practical DNA assay with greatly improved sensitivity. The detection limit of a model DNA target is 0.5 pM (3σ), which is about 3 orders of magnitude lower compared with traditional hybridization methods without HCR. Furthermore, the signal of complementary target can be clearly distinguished from that of single-base mismatched sequences, indicating the high specificity of the HCR-CBA. Moreover, this strategy is also successfully applied to the DNA analysis in complex biological samples, showing great potential in gene analysis and disease diagnosis in clinical samples.
authors.affiliation: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ren
authors.forename: Hongmei
authors.initials: H
authors.lastname: Liu
authors.forename: Wenxia
authors.initials: W
authors.lastname: Yang
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Fan
authors.forename: Lang
authors.initials: L
authors.lastname: Yang
authors.forename: Yucong
authors.initials: Y
authors.lastname: Wang
authors.forename: Chenghui
authors.initials: C
authors.lastname: Liu
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Li
mesh_terms: D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D057230:Limit of Detection; D059346:Magnets / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
chemical_list: D015342:DNA Probes; D001710:Biotin; D004247:DNA; D019809:Streptavidin
keywords: Bead assay; DNA analysis; Flow cytometry; Hybridization chain reaction; Signal amplification